The objective of this proposal is to identify the mechanisms involved in regulating gastrin-releasing peptide receptor (GRP-R) acute desensitization. The health-relatedness of this proposal stems from the fact that the GRP-R is expressed by and involved in the normal function of many tissues including those of the central nervous, immune, pulmonary, and gastroinestinal systems. The GRP-R also is avberrently expressed by, and can cause the proliferation of, a number of human cancers including those originating from the lung, breast, pancreas, stomach and colon. Therefore understanding the mechanisms whereby this receptor is turned off, or desensitized, consequent to agonist stimulation is of critical importance for understanding its functional regulation when expressed by normal and malignant cells. Most heptaspanning receptors studies are rapidly desensitized after agonist exposure, a phenomenon governed by their phosphorylation by 2nd messenger dependent and -independent kinases. In contrast we herein demonstrate that whereas the GRP-R is rapidly phosphorylated by both kinase types, phosphorylation does not influence its acute desensitization. Rather, we provide preliminary evidence in support of our novel hypothesis that acute G.P.-R desensitization is regulated by both the specific G proteins to which it couples and by its affinity for those G proteins. To evaluate this hypothesis, we will study the murine GRP-R natively expressed by Swiss 3T3 cells and ectopically expressed by Balb 3T3 cells. The benefit of these cell lines si that we show in this proposal that GRP-R bind only to Cxq/11 in Balb cells but to multiple G proteins in Swiss cells. Our specific aims then are to: 1) study GRP-R expressed by Balb cells to define its coupling to Galphaq/11; 2) identify the multiple G proteins activated by, and determine their binding sites to, GRP-R expressed by Swiss cells; and 3) identify the contribution various G proteins make to regulating acute GRP-R desensitization, and determine how this regulation is modulated by receptor-G protein binding affinity. We will achieve these aims by using site-directed mutagenesis to study the role of amino acid charge and hydrophobicity to regulating GRP-RGalphaq/11 coupling, and then study the acute desensitization response of various mutants in stably transfeted Balb 33 cells. We will identify the G proteins activated by GRP-R in Swiss cells using the non-hydrolyzable photo affinity GRP analog azidoanilido GTP (AAGTP). Using synthetic peptides to block G protein binding to the GRP-R along with AAGTP, we will define the specific site(s) each G protein uses to couple to this receptor. Finally, we will use synthetic peptides that block the activation of specific G proteins to determine the contribution of each to acute GRP-R desensitization in Swiss 3T3 cells.